Piperidine Compounds Useful as Malonyl-CoA Decarboxylase Inhibitors

ABSTRACT

The present invention provides methods for the use of compounds as depicted by structure I, pharmaceutical compositions containing the same, and methods for the prophylaxis, management and treatment of metabolic diseases and diseases modulated by MCD inhibition. The compounds disclosed in this invention are useful for the prophylaxis, management and treatment of diseases involving in malonyl-CoA regulated glucose/fatty acid metabolism pathway. In particular, these compounds and pharmaceutical composition containing the same are indicated in the prophylaxis, management and treatment of cardiovascular diseases, diabetes, cancer and obesity.

This application is a divisional application and claims the benefit ofpriority under 35 U.S.C. Section 120 of U.S. application Ser. No.10/900,705, filed on Jul. 28, 2004, which claims priority to U.S.Provisional Application Ser. No. 60/492,122, filed Aug. 1, 2003. Thedisclosure of each prior application is considered part of and isincorporated by reference in the disclosure of this application.

FIELD OF THE INVENTION

The present invention relates to methods of treatment of certainmetabolic diseases and the use of compounds and their prodrugs, and/orpharmaceutically acceptable salts, pharmaceutical compositionscontaining such compounds useful in treating such diseases. Inparticular, the invention relates to the use of compounds andcompositions for the prophylaxis, management or treatment ofcardiovascular diseases, diabetes, cancers, and obesity through theinhibition of malonyl-coenzyme A decarboxylase (malonyl-CoAdecarboxylase, MCD).

BACKGROUND OF THE INVENTION

Malonyl-CoA is an important metabolic intermediary produced by theenzyme Acetyl-CoA Carboxylase (ACC) in the body. In the liver,adipocytes, and other tissues, malonyl-CoA is a substrate for fatty acidsynthase (FAS). ACC and malonyl-CoA are found in skeletal muscle andcardiac muscle tissue, where fatty acid synthase levels are low. Theenzyme malonyl-CoA decarboxylase (MCD, EC 4.1.1.9) catalyzes theconversion of malonyl-CoA to acetyl-CoA and thereby regulatesmalonyl-CoA levels. MCD activity has been described in a wide array oforganisms, including prokaryotes, birds, and mammals. It has beenpurified from the bacteria Rhizobium trifolii (An et al., J. Biochem.Mol. Biol. 32:414-418 (1999)), the uropygial glands of waterfowl(Buckner, et al., Arch. Biochem. Biophys 177:539 (1976); Kim andKolattukudy Arch. Biochem. Biophys 190:585 (1978)), rat livermitochondria (Kim and Kolattukudy, Arch. Biochem. Biophys. 190:234(1978)), rat mammary glands (Kim and Kolattukudy, Biochim. Biophys, Acta531:187 (1978)), rat pancreatic β-cell (Voilley et al., Biochem. J.340:213 (1999)) and goose (Anseranser) (Jang et al., J. Biol. Chem.264:3500 (1989)). Identification of patients with MCD deficiency lead tothe cloning of a human gene homologous to goose and rat MCD genes (Gaoet al., J. Lipid. Res. 40:178 (1999); Sacksteder et al., J. Biol. Chem.274:24461 (1999); FitzPatrick et al., Am. J. Hum. Genet. 65:318 (1999)).A single human MCD mRNA is observed by Northern Blot analysis. Thehighest mRNA expression levels are found in muscle and heart tissues,followed by liver, kidney and pancreas, with detectable amounts in allother tissues examined.

Malonyl-CoA is a potent endogenous inhibitor of carnitinepalmitoyltransferase-I (CPT-I), an enzyme essential for the metabolismof long-chain fatty acids. CPT-I is the rate-limiting enzyme in fattyacid oxidation and catalyzes the formation of acyl-carnitine, which istransported from the cytosol across the mitochondrial membranes by acylcarnitine translocase. Inside of the mitochondria the long-chain fattyacids are transferred back to CoA form by a complementary enzyme,CPT-II, and, in the mitochondria, acyl-CoA enters the β-oxidationpathway generating acetyl-CoA. In the liver, high levels of acetyl-CoAoccurs for example following a meal, leading to elevated malonyl-CoAlevels, which inhibit CPT-I, thereby preventing fat metabolism andfavoring fat synthesis. Conversely, low malonyl-CoA levels favor fattyacid metabolism by allowing the transport of long-chain fatty acids intothe mitochondria. Hence, malonyl-CoA is a central metabolite that playsa key role in balancing fatty acid synthesis and fatty acid oxidation(Zammit, Biochem. J. 343:5050-515 (1999)). Recent work indicates thatMCD is able to regulate cytoplasmic as well as mitochondrial malonyl-CoAlevels [Alam and Saggerson, Biochem J. 334:233-241 (1998); Dyck et al.,Am J Physiology 275:H2122-2129 (1998)].

Although malonyl-CoA is present in muscle and cardiac tissues, only lowlevels of FAS have been detected in these tissues. It is believed thatthe role of malonyl-CoA and MCD in these tissues is to regulate fattyacid metabolism. This is achieved via malonyl-CoA inhibition of muscle(M) and liver (L) isoforms of CPT-I, which are encoded by distinct genes(McGarry and Brown, Eur. J. Biochem. 244:1-14 (1997)). The muscleisoform is more sensitive to malonyl-CoA inhibition (IC50 0.03 μM) thanthe liver isoform (IC₅₀ 2.5 μM). Malonyl-CoA regulation of CPT-I hasbeen described in the liver, heart, skeletal muscle and pancreaticβ-cells. In addition, malonyl-CoA sensitive acyl-CoA transferaseactivity present in microsomes, perhaps part of a system that deliversacyl groups into the endoplasmic reticulum, has also been described(Fraser et al., FEBS Lett. 446: 69-74 (1999)).

Cardiovascular Diseases: The healthy human heart utilizes availablemetabolic substrates. When blood glucose levels are high, uptake andmetabolism of glucose provide the major source of fuel for the heart. Inthe fasting state, lipids are provided by adipose tissues, and fattyacid uptake and metabolism in the heart down regulate glucosemetabolism. The regulation of intermediary metabolism by serum levels offatty acid and glucose comprises the glucose-fatty acid cycle (Randle etal., Lancet, 1:785-789 (1963)). Under ischemic conditions, limitedoxygen supply reduces both fatty acid and glucose oxidation and reducesthe amount of ATP produced by oxidative phosphorylation in the cardiactissues. In the absence of sufficient oxygen, glycolysis increases in anattempt to maintain ATP levels and a buildup of lactate and a drop inintracellular pH results. Energy is spent maintaining ion homeostasis,and myocyte cell death occurs as a result of abnormally low ATP levelsand disrupted cellular osmolarity. Additionally, AMPK, activated duringischemia, phosphorylates and thus inactivates ACC. Total cardiacmalonyl-CoA levels drop, CPT-I activity therefore is increased and fattyacid oxidation is favored over glucose oxidation. The beneficial effectsof metabolic modulators in cardiac tissue are the increased efficiencyof ATP/mole oxygen for glucose as compared to fatty acids and moreimportantly the increased coupling of glycolysis to glucose oxidationresulting in the net reduction of the proton burden in the ischemictissue.

A number of clinical and experimental studies indicate that shiftingenergy metabolism in the heart towards glucose oxidation is an effectiveapproach to decreasing the symptoms associated with cardiovasculardiseases, such as but not limited, to myocardial ischemia (Hearse,“Metabolic approaches to ischemic heart disease and its management”,Science Press). Several clinically proven anti-angina drugs includingperhexyline and amiodarone inhibit fatty acid oxidation via inhibitionof CPT-I (Kennedy et al., Biochem. Pharmacology, 52: 273 (1996)). Theantianginal drugs ranolazine, currently in Phase III clinical trials,and trimetazidine are shown to inhibit fatty acid β-oxidation (McCormacket al., Genet. Pharmac. 30:639 (1998), Pepine et al., Am. J. Cardiology84:46 (1999)). Trimetazidine has been shown to specifically inhibit thelong-chain 3-ketoactyl CoA thiolase, an essential step in fatty acidoxidation. (Kantor et al., Circ. Res. 86:580-588 (2000)).Dichloroacetate increases glucose oxidation by stimulating the pyruvatedehydrogenase complex and improves cardiac function in those patientswith coronary artery diseases (Wargovich et al., Am. J. Cardiol.61:65-70 (1996)). Inhibiting CPT-I activity through the increasedmalonyl-CoA levels with MCD inhibitors would result in not only a novel,but also a much safer method, as compared to other known small moleculeCPT-I inhibitors, to the prophylaxis and treatment of cardiovasculardiseases.

Most of the steps involved in glycerol-lipid synthesis occur on thecytosolic side of liver endoplasmic reticulum (ER) membrane. Thesynthesis of triacyl glycerol (TAG) targeted for secretion inside the ERfrom diacyl glycerol (DAG) and acyl CoA is dependent upon acyl CoAtransport across the ER membrane. This transport is dependent upon amalonyl-CoA sensitive acyl-CoA transferase activity (Zammit, Biochem. J.343: 505 (1999) Abo-Hashema, Biochem. 38: 15840 (1999) and Abo-Hashema,J. Biol. Chem. 274:35577 (1999)). Inhibition of TAG biosynthesis by aMCD inhibitor may improve the blood lipid profile and therefore reducethe risk factor for coronary artery disease of patients.

Diabetes: Two metabolic complications most commonly associated withdiabetes are hepatic overproduction of ketone bodies (in NIDDM) andorgan toxicity associated with sustained elevated levels of glucose.Inhibition of fatty acid oxidation can regulate blood-glucose levels andameliorate some symptoms of type II diabetes. Malonyl-CoA inhibition ofCPT-I is the most important regulatory mechanism that controls the rateof fatty acid oxidation during the onset of thehypoinsulinemic-hyperglucagonemic state. Several irreversible andreversible CPT-I inhibitors have been evaluated for their ability tocontrol blood glucose levels and they are all invariably hypoglycemic(Anderson, Current Pharmaceutical Design 4:1(1998)). A liver specificand reversible CPT-inhibitor, SDZ-CPI-975, significantly lowers glucoselevels in normal 18-hour-fasted nonhuman primates and rats withoutinducing cardiac hypertrophy (Deems et al., Am. J. Physiology 274:R524(1998)). Malonyl-CoA plays a significant role as a sensor of therelative availability of glucose and fatty acid in pancreatic β-cells,and thus links glucose metabolism to cellular energy status and insulinsecretion. It has been shown that insulin secretagogues elevatemalonyl-CoA concentration in β-cells (Prentki et al., Diabetes 45: 273(1996)). Treating diabetes directly with CPT-I inhibitors has, however,resulted in mechanism-based hepatic and myocardial toxicities. MCDinhibitors that inhibit CPT-I through the increase of its endogenousinhibitor, malonyl-CoA, are thus safer and superior as compared to CPT-Iinhibitors for treatment of diabetic diseases.

Cancers: Malonyl-CoA has been suggested to be a potential mediator ofcytotoxicity induced by fatty-acid synthase inhibition in human breastcancer cells and xenografts (Pizer et al., Cancer Res. 60:213 (2000)).It is found that inhibition of fatty acid synthase using antitumorantibiotic cerulenin or a synthetic analog C75 markedly increase themalonyl-CoA levels in breast carcinoma cells. On the other hand, thefatty acid synthesis inhibitor, TOFA (5-(tetradecyloxy)-2-furoic acid),which only inhibits at the acetyl-CoA carboxylase (ACC) level, does notshow any antitumor activity, while at the same time the malonyl-CoAlevel is decreased to 60% of the control. It is believed that theincreased malonyl-CoA level is responsible for the antitumor activity ofthese fatty acid synthase inhibitors. Regulating malonyl-CoA levelsusing MCD inhibitors thus constitutes a valuable therapeutic strategyfor the treatment of cancer diseases.

Obesity: It is suggested that malonyl-CoA may play a key role inappetite signaling in the brain via the inhibition of the neuropepetideY pathway (Loftus et al., Science 288: 2379 (2000)). Systemic orintracerebroventricular treatment of mice with fatty acid synthase (FAS)inhibitor cerulenin or C75 led to inhibition of feeding and dramaticweight loss. It is found that C75 inhibited expression of the prophagicsignal neuropeptide Y in the hypothalamus and acted in aleptin-independent manner that appears to be mediated by malonyl-CoA.Therefore control of malonyl-CoA levels through inhibition of MCDprovides a novel approach to the prophylaxis and treatment of obesity.

We have now found a novel use for compounds containing thiazoles andoxazoles, members of which are potent inhibitors of MCD. The compoundstested both in vitro and in vivo inhibit malonyl-CoA decarboxylaseactivities and increase the malonyl-CoA concentration in the animaltissues. In addition, by way of example, selected compounds induce asignificant increase in glucose oxidation as compared with the controlin an isolated perfused rat heart assay (McNeill, Measurement ofCardiovascular Function, CRC Press, 1997). Advantageously, preferredcompounds embodied in this application have more profound effects inmetabolism shift than the known metabolism modulators such as ranolazineor trimetazidine. The compounds useful for this invention andpharmaceutical compositions containing these compounds are thereforeuseful in medicine, especially in the prophylaxis, management andtreatment of various cardiovascular diseases, diabetes, cancers andobesity.

Additionally, these compounds are also useful as a diagnostic tool fordiseases associated with MCD deficiency or malfunctions.

SUMMARY OF THE INVENTION

The present invention provides methods for the use of compounds asdepicted by structure I, pharmaceutical compositions containing thesame, and methods for the prophylaxis, management and treatment ofmetabolic diseases and diseases modulated by MCD inhibition. Thecompounds disclosed in this invention are useful for the prophylaxis,management and treatment of diseases involving in malonyl-CoA regulatedglucose/fatty acid metabolism pathway. In particular, these compoundsand pharmaceutical composition containing the same are indicated in theprophylaxis, management and treatment of cardiovascular diseases,diabetes, cancer and obesity.

The present invention also includes within its scope diagnostic methodsfor the detection of diseases associated with MCD deficiency ormalfunctions.

The compounds useful in the present invention are represented by thefollowing structure:

Wherein R₁, R₂, R₃, n, X and Y are defined below. Also included withinthe scope of these compounds are the corresponding enantiomers,diastereoisomers, prodrugs, and pharmaceutically acceptable salts. Otheraspects of this invention will become apparent as the description ofthis invention continues. Hence, the foregoing merely summarizes certainaspects of the invention and is not intended, nor should it beconstrued, as limiting the invention in any way.

DETAILED DESCRIPTION OF THE INVENTION

The detailed description of the invention that follows is not intendedto be exhaustive or to limit the invention to the precise detailsdisclosed. It has been chosen and described to best explain the detailsof the invention to others skilled in the art.

The compounds useful in the present invention are represented by thefollowing formulae (I):

whereinR₁ is hydrogen, substituted C₁-C₁₂ alkyl, substituted C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkynyl, —COR₄, CONR₄R₅, —S(O)_(n)R₄, —SO₂NR₄R₅,—P(O)(R₅)₂, or —P(O)(R₅)₂;R₂ is selected from hydrogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,phenyl, substituted phenyl, aryl or heteroaryl;R₃ is selected from hydrogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,phenyl, substituted phenyl, aryl or heteroaryl;R₄ and R₅ are independently selected from hydrogen, C₁-C₁₂ alkyl,substituted C₁-C₁₂ alkyl, phenyl, substituted phenyl, aryl orheteroaryl;n is one or two;

X is C or N; Y is S or O;

its corresponding enantiomers, diastereoisomers or tautomers, or apharmaceutically acceptable salt, or a prodrug thereof in anpharmaceutically-acceptable carrier.

Preferably, the compounds in the present invention are represented bythe following formulae (Ia):

whereinR₁ is hydrogen, substituted C₁-C₁₂ alkyl, substituted C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkynyl, —COR₄, CONR₄R₅, —S(O)_(n)R₄, —SO₂NR₄R₅,—P(O)(R₅)₂, or —P(O)(R₅)₂;R₂ is selected from hydrogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,phenyl, substituted phenyl, aryl or heteroaryl;R₃ is selected from hydrogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,phenyl, substituted phenyl, aryl or heteroarylR₄ and R₅ are independently selected from hydrogen, C₁-C₁₂ alkyl,substituted C₁-C₁₂ alkyl, phenyl, substituted phenyl, aryl or heteroaryl

More preferably, the compounds in the present invention are representedby the general formulae Ia wherein

R₁ is —S(O)_(n)R₄;

R₂ is hydrogen;R₃ is C1-C6 alkyl or C1-C6 substituted alkyl.

Compositions

The compositions of the present invention comprise:

-   (a) a safe and therapeutically effective amount of an MCD inhibiting    compound I or II, its corresponding enantiomer, diastereoisomer or    tautomer, or pharmaceutically acceptable salt, or a prodrug thereof;    and-   (b) a pharmaceutically-acceptable carrier.

As discussed above, numerous diseases can be mediated by MCD relatedtherapy.

Accordingly, the compounds useful in this invention can be formulatedinto pharmaceutical compositions for use in prophylaxis, management andtreatment of these conditions. Standard pharmaceutical formulationtechniques are used, such as those disclosed in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

A “safe and therapeutically effective amount” of a compound useful inthe present invention is an amount that is effective, to inhibit MCD atthe site(s) of activity, in a subject, a tissue, or a cell, andpreferably in an animal, more preferably in a mammal, without undueadverse side effects (such as toxicity, irritation, or allergicresponse), commensurate with a reasonable benefit/risk ratio, when usedin the manner of this invention. The specific “safe and therapeuticallyeffective amount” will, obviously, vary with such factors as theparticular condition being treated, the physical condition of thepatient, the duration of treatment, the nature of concurrent therapy (ifany), the specific dosage form to be used, the carrier employed, thesolubility of the compound therein, and the dosage regimen desired forthe composition.

In addition to the selected compound useful for the present invention,the compositions of the present invention contain apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier”, as used herein, means one or morecompatible solid or liquid filler diluents or encapsulating substanceswhich are suitable for administration to a mammal. The term“compatible”, as used herein, means that the components of thecomposition are capable of being comingled with the subject compound,and with each other, in a manner such that there is no interaction whichwould substantially reduce the pharmaceutical efficacy of thecomposition under ordinary use situations. Pharmaceutically-acceptablecarriers must, of course, be of sufficiently high purity andsufficiently low toxicity to render them suitable for administrationpreferably to an animal, preferably mammal being treated.

Some examples of substances, which can serve aspharmaceutically-acceptable carriers or components thereof, are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents;flavoring agents; tableting agents, stabilizers; antioxidants;preservatives; pyrogen-free water; isotonic saline; and phosphate buffersolutions.

The choice of a pharmaceutically-acceptable carrier to be used inconjunction with the subject compound is basically determined by the waythe compound is to be administered.

If the subject compound is to be injected, the preferredpharmaceutically-acceptable carrier is sterile, physiological saline,with blood-compatible suspending agent, the pH of which has beenadjusted to about 7.4. In particular, pharmaceutically-acceptablecarriers for systemic administration include sugars, starches, celluloseand its derivatives, malt, gelatin, talc, calcium sulfate, vegetableoils, synthetic oils, polyols, alginic acid, phosphate buffer solutions,emulsifiers, isotonic saline, and pyrogen-free water. Preferred carriersfor parenteral administration include propylene glycol, ethyl oleate,pyrrolidone, ethanol, and sesame oil. Preferably, thepharmaceutically-acceptable carrier, in compositions for parenteraladministration, comprises at least about 90% by weight of the totalcomposition.

The compositions of this invention are preferably provided in unitdosage form. As used herein, a “unit dosage form” is a composition ofthis invention containing an amount of a compound that is suitable foradministration to an animal, preferably mammal subject, in a singledose, according to good medical practice. (The preparation of a singleor unit dosage form however, does not imply that the dosage form isadministered once per day or once per course of therapy. Such dosageforms are contemplated to be administered once, twice, thrice or moreper day, and are expected to be given more than once during a course oftherapy, though a single administration is not specifically excluded.The skilled artisan will recognize that the formulation does notspecifically contemplate the entire course of therapy and such decisionsare left for those skilled in the art of treatment rather thanformulation.) These compositions preferably contain from about 5 mg(milligrams), more preferably from about 10 mg to about 1000 mg, morepreferably to about 500 mg, most preferably to about 300 mg, of theselected compound.

The compositions useful for this invention may be in any of a variety offorms, suitable (for example) for oral, nasal, rectal, topical(including transdermal), ocular, intracereberally, intravenous,intramuscular, or parenteral administration. (The skilled artisan willappreciate that oral and nasal compositions comprise compositions thatare administered by inhalation, and made using available methodologies.)Depending upon the particular route of administration desired, a varietyof pharmaceutically-acceptable carriers well-known in the art may beused. These include solid or liquid fillers, diluents, hydrotropies,surface-active agents, and encapsulating substances. Optionalpharmaceutically-active materials may be included, which do notsubstantially interfere with the inhibitory activity of the compound.The amount of carrier employed in conjunction with the compound issufficient to provide a practical quantity of material foradministration per unit dose of the compound. Techniques andcompositions for making dosage forms useful in the methods of thisinvention are described in the following references, all incorporated byreference herein: Modern Pharmaceutics, Chapters 9 and 10 (Banker &Rhodes, editors, 1979); Lieberman et al., Pharmaceutical Dosage Forms:Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms2d Edition (1976).

Various oral dosage forms can be used, including such solid forms astablets, capsules, granules and bulk powders. These oral forms comprisea safe and effective amount, usually at least about 5%, and preferablyfrom about 25% to about 50%, of the compound. Tablets can be compressed,tablet triturates, enteric-coated, sugar-coated, film-coated, ormultiple-compressed, containing suitable binders, lubricants, diluents,disintegrating agents, coloring agents, flavoring agents, flow-inducingagents, and melting agents. Liquid oral dosage forms include aqueoussolutions, emulsions, suspensions, solutions and/or suspensionsreconstituted from non-effervescent granules, and effervescentpreparations reconstituted from effervescent granules, containingsuitable solvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, melting agents, coloring agents and flavoringagents.

The pharmaceutically-acceptable carrier suitable for the preparation ofunit dosage forms for peroral administration are well-known in the art.Tablets typically comprise conventional pharmaceutically-compatibleadjuvants as inert diluents, such as calcium carbonate, sodiumcarbonate, mannitol, lactose and cellulose; binders such as starch,gelatin and sucrose; disintegrants such as starch, alginic acid andcroscarmelose; lubricants such as magnesium stearate, stearic acid andtalc. Glidants such as silicon dioxide can be used to improve flowcharacteristics of the powder mixture. Coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. The selection of carriercomponents depends on secondary considerations like taste, cost, andshelf stability, which are not critical for the purposes of the subjectinvention, and can be readily made by a person skilled in the art.

Peroral compositions also include liquid solutions, emulsions,suspensions, and the like. The pharmaceutically-acceptable carrierssuitable for preparation of such compositions are well known in the art.Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, AVICEL RC-591, tragacanth and sodium alginate; typicalwetting agents include lecithin and polysorbate 80; and typicalpreservatives include methyl paraben and sodium benzoate. Peroral liquidcompositions may also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

Such compositions may also be coated by conventional methods, typicallywith pH or time-dependent coatings, such that the subject compound isreleased in the gastrointestinal tract in the vicinity of the desiredtopical application, or at various times to extend the desired action.Such dosage forms typically include, but are not limited to, one or moreof cellulose acetate phthalate, polyvinylacetate phthalate,hydroxypropyl methyl cellulose phthalate, ethyl cellulose, Eudragitcoatings, waxes and shellac.

Compositions of the subject invention may optionally include other drugactives.

Other compositions useful for attaining systemic delivery of the subjectcompounds include sublingual, buccal and nasal dosage forms. Suchcompositions typically comprise one or more of soluble filler substancessuch as sucrose, sorbitol and mannitol; and binders such as acacia,microcrystalline cellulose, carboxymethyl cellulose and hydroxypropylmethyl cellulose. Glidants, lubricants, sweeteners, colorants,antioxidants and flavoring agents disclosed above may also be included.

The compositions of this invention can also be administered topically toa subject, e.g., by the direct application or spreading of thecomposition on the epidermal or epithelial tissue of the subject, ortransdermally via a “patch”. Such compositions include, for example,lotions, creams, solutions, gels and solids. These topical compositionspreferably comprise a safe and effective amount, usually at least about0.1%, and preferably from about 1% to about 5%, of the compound.Suitable carriers for topical administration preferably remain in placeon the skin as a continuous film, and resist being removed byperspiration or immersion in water. Generally, the carrier is organic innature and capable of having dispersed or dissolved therein thecompound. The carrier may include pharmaceutically-acceptable emollient,emulsifiers, thickening agents, solvents and the like.

Methods of Administration

The compounds and compositions useful in this invention can beadministered topically or systemically. Systemic application includesany method of introducing compound into the tissues of the body, e.g.,intra-articular, intrathecal, epidural, intramuscular, transdermal,intravenous, intraperitoneal, subcutaneous, sublingual administration,inhalation, rectal, or oral administration. The compounds useful in thepresent invention are preferably administered orally.

The specific dosage of the compound to be administered, as well as theduration of treatment is to be individualised by the treatingclinicians. Typically, for a human adult (weighing approximately 70kilograms), from about 5 mg, preferably from about 10 mg to about 3000mg, more preferably to about 1000 mg, more preferably to about 300 mg,of the selected compound is administered per day. It is understood thatthese dosage ranges are by way of example only, and that dailyadministration can be adjusted depending on the factors listed above.

In all of the foregoing, of course, the compounds useful in the presentinvention can be administered alone or as mixtures, and the compositionsmay further include additional drugs or excipients as appropriate forthe indication. For example, in the treatment of cardiovasculardiseases, it is clearly contemplated that the invention may be used inconjunction with beta-blockers, calcium antagonists, ACE inhibitors,diuretics, angiotensin receptor inhibitors, or known cardiovasculardrugs or therapies. Hence, in this example, compounds or compositionsuseful in this invention are useful when dosed together with anotheractive and can be combined in a single dosage form or composition.

These compositions can also be administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles, and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine, orphosphatidylcholines.

DEFINITIONS

As used herein, “alkyl” means a straight chain alkane, alkene, or alkynesubstituent containing only carbon and hydrogen, such as methyl, ethyl,butyl, pentyl, heptyl and the like. Alkyl groups can be saturated orunsaturated (i.e., containing —C═C— or —C≡C— linkages), at one orseveral positions. When a specific degree of unsaturation is preferred,said substituent is referred to as either “alkenyl” or “alkynyl”,denoting substituents containing —C═C— or —C≡C— linkages, respectively.The number of carbons may be denoted as “C_(i)-C_(j)-alkyl” wherein Iand j refer to the minimum and maximum number of carbon atoms,respectively. Typically, alkyl groups will comprise 1 to 12 carbonatoms, preferably 1 to 10, and more preferably 2 to 8 carbon atoms.

As used herein, “substituted alkyl” means a hydrocarbon substituent,which is linear, cyclic or branched, in which one or more hydrogen atomsare substituted by carboxy, hydroxy, alkoxy, cyano, nitro, carbonyl,aryl, carboxyalkyl, mercapto, amino, amido, ureido, carbamoyl,sulfonamido, sulfamido, or halogen. Preferred substituted alkyls havetheir alkyl spacers (i.e., portion which is alkyl) of 1 to about 5carbons, and may be branched or linear, and may include cyclicsubstituents, either as part or all of their structure. Preferredexamples of “substituted alkyls” include 4-carboxybutyl,pyridin-2-ylmethyl, and 1,3-thiazol-2-ylmethyl, benzyl, phenethyl, andtrifluoromethyl. The term “substituted alkyl” may be combined with otherart accepted terms. For example “substituted alkoxy” means alkoxy asunderstood in the art, wherein the alkyl portion of the substituent issubstituted.

As used herein, “branched alkyl” means a subset of “alkyl”, and thus isa hydrocarbon substituent, which is branched. Preferred branched alkylsare of 3 to about 12 carbons, and may include cycloalkyl within theirstructure. Examples of branched alkyl include isopropyl, isobutyl,1,2-dimethyl-propyl, cyclopentylmethyl and the like. The term “branchedalkyl” may be combined with other art accepted terms. For example“branched alkoxy” means alkoxy as understood in the art, wherein thealkyl portion of the substituent is branched.

As used herein, “cycloalkyl” is a hydrocarbon substituent that iscyclic, and can be substituted or unsubstituted. Where it issubstituted, one or more hydrogen atoms are substituted by carboxy,hydroxy, alkoxy, cyano, nitro, carbonyl, aryl, carboxyalkyl, mercapto,amino, amido, ureido, carbamoyl, sulfonamido, sulfamido, or halogen.Preferred cyclic alkyls are of 3 to about 7 carbons. Examples ofcycloalkyl include cyclopropyl, cyclopentyl, 4-fluoro-cyclohexyl,2,3-dihydroxy-cyclopentyl, and the like.

As used herein, “alkylene” is an alkyl diradical, i.e., an alkyl thathas open valences on two different carbon atoms. Hence “(alkylene)R_(i)”is an alkyl diradical attached at one carbon and having substituentR_(i) attached at another carbon, which may be one or more carbons awayfrom the point of attachment. Alkylene can be linear, branched, orcyclic. Examples of alkylene include —CH₂—, CH₂CH₂—, —(CH₂)₄—,-(cyclohexyl)-, and the like.

As used herein, “aryl” is a substituted or unsubstituted aromatic, i.e.,Hückel 4n+2 rule applies, radical having a single-ring (e.g., phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl), which may containzero to 4 heteroatoms. Hence the term “heteroaryl” is clearlycontemplated in the term “aryl”. Preferred carbocyclic aryl, is phenyl.Preferred monocyclic heterocycles, i.e., heteroaryls, are 5 or 6membered rings. Preferably, where the term “aryl” represents an aromaticheterocycle, it is referred to as “heteroaryl” or “heteroaromatic”, andhas one or more heteroatom(s). Preferred numbers of such heteroatoms arefrom one to three N atoms, and preferably when “heteroaryl” is aheterocycle of five members, it has one or two heteroatoms selected fromO, N, or S. Hence, preferred heterocycles have up to three, morepreferably two or less, heteroatoms present in the aromatic ring. Theskilled artisan will recognize that among heteroaryl, there are bothfive and six membered rings. Examples of “heteroaryl” include; thienyl,pyridyl, pyrimidyl, pyridazyl, furyl, oxazolyl, imidazolyl, thiazolyl,oxadiazilyl, triazinyl, triazolyl, thiadiazolyl, and others, which theskilled artisan will recognize. In this definition it is clearlycontemplated that substitution on the aryl ring is within the scope ofthis invention. Where substitution occurs, the radical is referred to as“substituted aryl”. Preferably one to three, more preferably one or two,and most preferably one substituent is attached to the aryl ring.Although many substituents will be useful, preferred substituentsinclude those commonly found in aryl compounds, such as alkyl, hydroxy,alkoxy, cyano, nitro, halo, haloalkyl, mercapto and the like. Suchsubstituents are prepared using known methodologies. These substituentsmay be attached at various positions of the aryl ring, and wherein agiven placement is preferred, such placement is indicated by“o,m,p-R_(i)-aryl”. Thus, if substituent R_(i) is attached at the paraposition of the aryl, then this is indicated as “p-R_(i)-substitutedaryl”.

As used herein, “amide” includes both RNR′CO— (in the case of R=alkyl,alkaminocarbonyl-) and RCONR′— (in the case of R=alkyl, alkylcarbonylamino-).

As used herein, “ester” includes both ROCO— (in the case of R=alkyl,alkoxycarbonyl-) and RCOO— (in the case of R=alkyl, alkylcarbonyloxy-).

As used herein, “halogen” is a chloro, bromo, fluoro or iodo atomradical. Chloro, bromo and fluoro are preferred halogens. The term“halogen” also contemplates terms sometimes referred to as “halo” or“halide”.

As used herein, “alkylamino” is an amine radical in which at least onehydrogen atom on the nitrogen has been replaced with alkyl. Preferredexamples include ethylamino, butylamino, isopropylamino, and the like.The alkyl component may be linear, branched, cyclic, substituted,saturated, or unsaturated.

As used herein, “alkylsulfanyl” is a thiol radical in which the hydrogenatom on sulfur has been replaced with alkyl. Preferred examples includeethylsulfanyl, butylsulfanyl, isopropylsulfanyl, and the like. The alkylcomponent may be linear, branched, cyclic, substituted, saturated, orunsaturated.

As used herein, “alkoxy” is a hydroxyl radical in which the hydrogenatom on oxygen has been replaced with alkyl. Preferred examples includeethoxy, butoxy, benzyloxy, and the like. The alkyl component may belinear, branched, cyclic, substituted, saturated, or unsaturated.

As used herein, “heterocycle(s)” means ring systems, preferably of 3-7members, which are saturated or unsaturated, and non-aromatic. These maybe substituted or unsubstituted, and are attached to other parts of themolecule via any available valence, preferably any available carbon ornitrogen. More preferred heterocycles are of 5 or 6 members. Insix-membered monocyclic heterocycles, the heteroatom(s) are from one tothree of O, S, or N, and wherein when the heterocycle is five-membered,preferably it has one or two heteroatoms selected from O, N, or S.

As used herein, “heterocyclyl” means radical heterocycles. These may besubstituted or unsubstituted, and are attached to other via anyavailable valence, preferably any available carbon or nitrogen.

As used herein, “sulfamido” means an alkyl-N—S(O)₂N—, aryl-NS(O)₂N— orheterocyclyl-NS(O)₂N— group wherein the alkyl, aryl or heterocyclylgroup is as defined herein above.

As used herein, “sulfonamido” means an alkyl-S(O)₂N—, aryl-S(O)₂N— orheterocyclyl-S(O)₂N— group wherein the alkyl, aryl or heterocyclcylgroup is as herein described.

As used herein, “ureido” means an alkyl-NCON—, aryl-NCON— orheterocyclyl-NCON— group wherein the alkyl, aryl or heterocyclyl groupis as herein described.

A substituent referred to as a radical in this specification may form aring with another radical as described herein. When such radicals arecombined, the skilled artisan will understand that there are nounsatisfied valences in such a case, but that specific substitutions,for example a bond for a hydrogen, is made. Hence certain radicals canbe described as forming rings together. The skilled artisan willrecognize that such rings can and are readily formed by routine chemicalreactions, and it is within the purview of the skilled artisan to bothenvision such rings and the methods of their formations. Preferred arerings having from 3-7 members, more preferably 5 or 6 members. Compoundsdescribed herein may have cyclic structures therein, such as a ring R₁and R₂. In that regard the skilled artisan recognizes that this methodof description is routine in medicinal chemistry, though such may notrigorously reflect the chemical synthetic route. As used herein the term“ring” or “rings” when formed by the combination of two radicals refersto heterocyclic or carbocyclic radicals, and such radicals may besaturated, unsaturated, or aromatic. For example, preferred heterocyclicring systems include heterocyclic rings, such as morpholinyl,piperidinyl, imidazolyl, pyrrolidinyl, and pyridyl.

The skilled artisan will recognize that the radical of formula:

represents a number of different functionalities. Preferredfunctionalities represented by this structure include amides, ureas,thioureas, carbamates, esters, thioesters, amidines, ketones, oximes,nitroolefines, hydroxyguanidines and guanidines. More preferredfunctionalities include ureas, thioureas, amides, and carbamates.

The skilled artisan will recognize that some structures described hereinmay be resonance forms or tautomers of compounds that may be fairlyrepresented by other chemical structures. The artisan recognizes thatsuch structures are clearly contemplated within the scope of thisinvention, although such resonance forms or tautomers are notrepresented herein. For example, the structures:

clearly represent the same compound(s), and reference to either clearlycontemplates the other. In addition, the compounds useful in thisinvention can be provided as prodrugs, the following of which serve asexamples:

wherein R is a group (or linkage) removed by biological processes.Hence, clearly contemplated in this invention is the use compoundsprovided as biohydrolyzable prodrugs, as they are understood in the art.“Prodrug”, as used herein is any compound wherein when it is exposed tothe biological processes in an organism, is hydrolyzed, metabolized,derivatized or the like, to yield an active substance having the desiredactivity. The skilled artisan will recognize that prodrugs may or maynot have any activity as prodrugs. It is the intent that the prodrugsdescribed herein have no deleterious effect on the subject to be treatedwhen dosed in safe and effective amounts. These include for example,biohydrolyzable amides and esters. A “biohydrolyzable amide” is an amidecompound which does not essentially interfere with the activity of thecompound, or that is readily converted in vivo by a cell, tissue, orhuman, mammal, or animal subject to yield an active compound. A“biohydrolyzable ester” refers to an ester compound that does notinterfere with the activity of these compounds or that is readilyconverted by an animal to yield an active compound. Such biohydrolyzableprodrugs are understood by the skilled artisan and are embodied inregulatory guidelines.

Compounds and compositions herein also specifically contemplatepharmaceutically acceptable salts, whether cationic or anionic. A“pharmaceutically-acceptable salt” is an anionic salt formed at anyacidic (e.g., carboxyl) group, or a cationic salt formed at any basic(e.g., amino) group. Many such salts are known in the art, as describedin World Patent Publication 87/05297, Johnston et al., published Sep.11, 1987 (incorporated by reference herein). Preferred counter-ions ofsalts formable at acidic groups can include cations of salts, such asthe alkali metal salts (such as sodium and potassium), and alkalineearth metal salts (such as magnesium and calcium) and organic salts.Preferred salts formable at basic sites include anions such as thehalides (such as chloride salts). Of course, the skilled artisan isaware that a great number and variation of salts may be used, andexamples exist in the literature of either organic or inorganic saltsuseful in this manner.

Inasmuch as the compounds useful in this invention may contain one ormore stereogenic centers, “Optical isomer”, “stereoisomer”,“enantiomer,” “diastereomer,” as referred to herein have the standardart recognized meanings (cf. Hawleys Condensed Chemical Dictionary, 11thEd.) and are included in these compounds, whether as racemates, or theiroptical isomers, stereoisomers, enantiomers, and diastereomers.

As used herein, the term “metabolic disease”, means a group ofidentified disorders in which errors of metabolism, imbalances inmetabolism, or sub-optimal metabolism occur. The metabolic diseases asused herein also contemplate a disease that can be treated through themodulation of metabolism, although the disease itself may or may not becaused by specific metabolism blockage. Preferably, such metabolicdisease involves glucose and fatty acid oxidation pathway. Morepreferably, such metabolic disease involves MCD or is modulated bylevels of Malonyl CoA, and is referred to herein as an “MCD or MCArelated disorder.”

Preparation of Compounds Useful in this Invention

The starting materials used in preparing the compounds useful in thisinvention are known, made by known methods, or are commerciallyavailable. It will be apparent to the skilled artisan that methods forpreparing precursors and functionality related to the compounds claimedherein are generally described in the literature. The skilled artisangiven the literature and this disclosure is well equipped to prepare anyof these compounds.

It is recognized that the skilled artisan in the art of organicchemistry can readily carry out manipulations without further direction,that is, it is well within the scope and practice of the skilled artisanto carry out these manipulations. These include reduction of carbonylcompounds to their corresponding alcohols, reductive alkylation ofamines, oxidations, acylations, aromatic substitutions, bothelectrophilic and nucleophilic, etherifications, esterification,saponification and the like. These manipulations are discussed instandard texts such as March Advanced Organic Chemistry (Wiley), Careyand Sundberg, Advanced Organic Chemistry and the like.

The skilled artisan will readily appreciate that certain reactions arebest carried out when other functionality is masked or protected in themolecule, thus avoiding any undesirable side reactions and/or increasingthe yield of the reaction. Often the skilled artisan utilizes protectinggroups to accomplish such increased yields or to avoid the undesiredreactions. These reactions are found in the literature and are also wellwithin the scope of the skilled artisan. Examples of many of thesemanipulations can be found for example in T. Greene and P. WutsProtecting Groups in Organic Synthesis, 2^(nd) Ed., John Wiley & Sons(1991).

The following example schemes are provided for the guidance of thereader, and represent preferred methods for making the compoundsexemplified herein. These methods are not limiting, and it will beapparent that other routes may be employed to prepare these compounds.Such methods specifically include solid phase based chemistries,including combinatorial chemistry. The skilled artisan is thoroughlyequipped to prepare these compounds by those methods given theliterature and this disclosure.

As shown in the above scheme, 4-(N-protected amino)-piperidine (1) or2-(N-protected amino)-pyrrolidine compounds were reacted with halides(acyl chloride, sulfonyl chlorides, alkyl halides etc) or isocyanates toprovide desired amides, sulfonamides, amines or ureas (2) in good yield.Removal of the protecting group at C-4 position gave rise to the desired4-aminopiperidine derivatives (3).

In Vitro MCD Inhibitory Assay

The conversion of acetyl-CoA from malonyl-CoA was assayed using amodified protocol as previously described by Kim, Y. S. and Kolattukudy,P. E. in 1978 (Arch. Biochem. Biophys 190:585 (1978)). As shown in eq.1-3, the establishment of the kinetic equilibrium between malate/NAD andoxaloacetate/NADH was catalyzed by malic dehydrogenase (eq. 2). Theenzymatic reaction product of MCD, acetyl-CoA, shifted the equilibriumby condensing with oxaloacetate in the presence of citrate synthase (eq.3), which resulted in a continuous generation of NADH from NAD. Theaccumulation of NADH can be continuously followed by monitoring theincrease of fluorescence emission at 460 nm on a fluorescence platereader. The fluorescence plate reader was calibrated using the authenticacetyl-CoA from Sigma. For a typical 96-well plate assay, the increasein the fluorescence emission (λ_(ex)=360 nm, λ_(em)=460 nm, for NADH) ineach well was used to calculate the initial velocity of hMCD. Each 50 μLassay contained 10 mM phosphate buffered saline (Sigma), pH 7.4, 0.05%Tween-20, 25 mM K₂HPO₄—KH₂PO₄ (Sigma), 2 mM Malate (Sigma), 2 mM NAD(Boehringer Mannheim), 0.786 units of MD (Roche Chemicals), 0.028 unitof CS (Roche Chemicals), 5-10 nM hMCD, and varying amounts of MCAsubstrate. Assays were initiated by the addition of MCA, and the rateswere corrected for the background rate determined in the absence ofhMCD.

Isolated Working Rat Heart Assay Protocol

Isolated working hearts from male Sprague-Dawley rats (300-350 g) aresubjected to a 60-minute aerobic perfusion period. The working heartsare perfused with 95% O₂, 5% CO₂ with a modified Krebs-Henseleitsolution containing 5 mM glucose; 100 μU/mL insulin; 3% fatty acid-freeBSA; 2.5 mM free Ca²⁺, and 0.4 to 1.2 mmol/L palmitate (Kantor et al.,Circulation Research 86:580-588 (2000)). The test compound is added 5minutes before the perfusion period. DMSO (0.05%) is used as control.

Measurement of Glucose Oxidation Rates

Samples were taken at 10-minute intervals for measurements ofexperimental parameters. Glucose oxidation rates are determined by thequantitative collection of ¹⁴CO₂ produced by hearts perfused with buffercontaining [U14]-Glucose (R. Barr and G. Lopaschuk, in “Measurement ofcardiovascular function”, McNeill, J. H. ed., Chapter 2, CRC press, NewYork (1997)). After the perfusion, the ¹⁴CO₂ from the perfusae issubsequently released by injecting 1 mL of perfusate into sealed testtube containing 1 mL of 9NH₂SO4. The tube was sealed with a rubberstopper attached to a scintillation vial containing a piece of filterpapers saturated with 300 μl of hyamine hydroxide. The scintillationvials with filter papers were then removed and Ecolite ScintillationFluid added. Samples were counted by standard procedures as describedabove. Average rates of glucose oxidation for each phase of perfusionare expressed as μmol/min/g dry wt as described above.

Measurement of Fatty Acid Oxidation Rates:

Rates of fatty acid oxidation are determined using the same method asdescribed above for glucose oxidation rate measurement using[¹⁴C]palmitate or by the quantitative collection of ³H₂O produced byhearts perfused with buffer containing [5-³H]palmitate (R. Barr and G.Lopaschuk, in “Measurement of cardiovascular function”, McNeill, J. H.ed., Chapter 2, CRC press, New York (1997)). ³H₂O was separated from[5⁻³H]palmitate by treating 0.5 mL buffer samples with 1.88 mL of amixture of chloroform/methanol (1:2 v:v) and then adding 0.625 mL ofchloroform and 0.625 mL of a 2 M KCl/HCl solution. The sample iscentrifuged for 10 min and aqueous phase was removed and treated with amixture of 1 mL of chloroform, 1 mL of methanol and 0.9 mL KCl/HCl witha ration of 1:1:0.9. The aqueous layer was then counted for total ³H₂Odetermination. This process resulted in greater than 99.7% extractionand separation of ³H₂O from the pamiltate. Average rates of fatty acidoxidation for each phase of perfusion are expressed as nmol/min/g dry wtafter taking consideration the dilution factor.

Active compounds are characterized by an increase in glucose oxidationand/or decrease in fatty acid oxidation as compared to the controlexperiments (DMSO). The compounds that caused statistically significantincreases in glucose oxidation and/or decrease in fatty acid oxidationare considered to be active. Statistical significance was calculatedusing the Student's t test for paired or unpaired samples, asappropriate. The results with P<0.05 are considered to be statisticallysignificant.

EXAMPLES

To further illustrate this invention, the following examples areincluded. The examples should not be construed as specifically limitingthe invention. Variations of these examples within the scope of theclaims are within the purview of one skilled in the art are consideredto fall within the scope of the invention as described, and claimedherein. The reader will recognize that the skilled artisan, armed withthe present disclosure, and skill in the art is able to prepare and usethe invention without exhaustive examples.

Trademarks used herein are examples only and reflect illustrativematerials used at the time of the invention. The skilled artisan willrecognize that variations in lot, manufacturing processes, and the like,are expected. Hence the examples, and the trademarks used in them arenon-limiting, and they are not intended to be limiting, but are merelyan illustration of how a skilled artisan may choose to perform one ormore of the embodiments of the invention.

¹H nuclear magnetic resonance spectra (NMR) is measured in CDCl₃ orother solvents as indicated by a Varian NMR spectrometer (Unity Plus400, 400 MHz for ¹H) unless otherwise indicated and peak positions areexpressed in parts per million (ppm) downfield from tetramethylsilane.The peak shapes are denoted as follows, s, singlet; d, doublet; t,triplet; q, quartet; m, multiplet.

The following abbreviations have the indicated meanings:

-   -   Ac=acetyl    -   Bn=benzyl    -   Bz=benzoyl    -   CDI=carbonyl diimidazole    -   CH₂Cl₂=dichloromethane    -   DIBAL=diisobutylaluminum hydride    -   DMAP=4-(dimethylamino)-pyridine    -   DMF=N,N-dimethylformamide    -   DMSO=dimethylsulfoxide    -   EDCI or ECAC=1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide        hydrochloric acid    -   ESIMS=electron spray mass spectrometry    -   Et₃N=triethylamine    -   EtOAc=ethyl acetate    -   HMTA=hexamethylenetetramine    -   LDA=lithium diisopropylamide    -   LHDMS=lithium bis(trimethylsilyl)amide    -   MgSO₄=magnesium sulfate    -   NaH=sodium hydride    -   NBS=N-bromosuccinimide    -   NCS=N-chlorosuccinimide    -   NH₄Cl=ammonium chloride    -   Ph=phenyl    -   Py=pyridinyl    -   r.t.=room temperature    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran    -   TLC=thin layer chromatography    -   Tf₂O=triflic anhydride    -   Alkyl group abbreviations    -   Me=methyl    -   Et=ethyl    -   n-Pr=normal propyl    -   i-Pr=isopropyl    -   n-Bu=normal butyl    -   i-Bu=isobutyl    -   t-Bu=tertiary butyl    -   s-Bu=secondary butyl    -   c-Hex=cyclohexyl

Example 1 Preparation of1-Isopropyl-1-(2-mercapto-benzothiazol-6-yl)-3-[1-(4,4,4-trifluoro-butyl)-piperidin-4-yl]-urea

Step 1:

4-(N-BOC-amino)-piperidine (400 mg, 2.00 mmol), 1-bromo-4-methylpentane(436 μl, 3.00 mmol), and potassium carbonate (415 mg, 3.00 mmol) werecombined in DMF (8 ml) and stirred at 100° C. for 4 hours. The reactionmixture was cooled and taken up in ethyl acetate and washed with water,then brine. After drying over magnesium sulfate, the organic phase wasconcentrated in vacuo. The resulting solid was dissolved in 1:1dichlormethane/trifluoroacetic acid (4 ml) and stirred at ambienttemperature overnight. The solution was concentrated in vacuo, taken upin ethyl acetate, and stirred with an excess of resin boundbenzyltrimethylammonium bicarbonate for 3 hours. Filtration andconcentration yielded 199 mg (54%). ¹H NMR (CD₃OD) δ 0.90 (d, 6H), 1.22(m, 2H), 1.58 (m, 1H), 1.72 (m, 2H), 1.89 (m, 2H), 2.22 (app d, 2H),3.05 (m, 4H), 3.41 (m, 1H), 3.65 (d, 2H).

Step 2:

4-amino-N-(4-methylpentyl)-piperidine (199 mg, 1.08 mmol) andN,N-diisopropyl-ethylamine (189 μl, 1.08 mmol) were dissolved in 1:1dichloromethane/ethyl acetate (5 ml) and cooled to 0° C. Triphosgene(107 mg, 0.36 mmol) was added and the mixture was stirred half hours.The solvent was evaporated and the intermediate dissolved in THF (5 ml)with 6-isopropylamino-1,3-benzothiazole-2-thiol (242 mg, 1.08 mmol) andheated at 60° C. overnight. Solvent was evaporated and the crude productpurified by preparative TLC (10% methanol, 90% dichloromethane) to yield31 mg (7%). ¹H NMR (DMSO-d₆) δ 0.92 (d, 6H), 0.95 (d, 6H), 1.04 (q, 2H),1.45 (m, 4H), 1.62 (d, 2H), 2.92 (m, 2H), 2.36 (m, 2H), 3.25 (d, 2H),4.58 (m, 1H), 4.84 (d, 1H), 6.95 (d, 1H), 7.24 (d, 1H), 7.32 (s, 1H);ESIMS: m/z 435 (M+H).

Example 2 Preparation of1-Isopropyl-1-(2-mercapto-benzothiazol-6-yl)-3-[1-(propane-2-sulfonyl)-piperidin-4-yl]-urea

Step 1:

4-(N-BOC-amino)-piperidine (200 mg, 1.00 mmol) and triethylamine (139μl, 1.00 mmol) were combined in dichloromethane (5 ml) and cooled to−10° C. Isopropylsulfonyl chloride (139 μl, 1.00 mmol) was added and thereaction mixture stirred at ambient temperature overnight. The reactionmixture was concentrated in vacuo and the resulting solid was dissolvedin 1:1 dichlormethane/trifluoroacetic acid (5 ml) and stirred for 30minutes. The solution was concentrated in vacuo, taken up in ethylacetate, and extracted with aqueous sodium bicarbonate solution, waterand brine. The organic layer was dried over magnesium sulfate andconcentrated to yield 179 mg (81%). ¹H NMR (CD₃OD) δ 1.27 (d, 6H), 1.58(m, 2H), 2.04 (m, 2H), 3.01 (t, 2H), 3.24 (m, 1H), 3.43 (m, 1H), 3.70(d, 2H).

Step 2:

4-amino-N-(isopropylsulfanyl)-piperidine (179 mg, 0.805 mmol) andN,N-diisopropylethylamine (140 μl, 0.805 mmol) were dissolved indichloromethane (3 ml) and cooled to 0° C. Triphosgene (80 mg, 0.268mmol) was added and the mixture was stirred 20 minutes. The solvent wasevaporated and the intermediate dissolved in THF (3 ml) with6-isopropylamino-1,3-benzothiazole-2-thiol (90 mg, 0.403 mmol) andheated at 60° C. overnight. The product was purified by preparative TLC(20% acetonitrile, 80% dichloromethane) to yield 16.5 mg (4%). ¹H NMR(CDCl₃) δ1.01 (d, 6H), 1.22 (d, 6H), 1.89 (d, 2H), 2.96 (t, 2H), 3.16(m, 1H), 3.70 (m, 4H), 4.84 (m, 1H), 7.14 (d, 1H), 7.22 (s, 1H), 7.39(d, 1H); ESIMS: m/z 457 (M+H).

Example 3 Preparation of1-isopropyl-1-(2-mercapto-benzothiazole-6-yl)-3-[1-(4-methyl-pentanolyl)-piperidin-4-yl]-urea

Step 1:

4-(N-BOC-amino)-piperidine (200 mg, 1.00 mmol) and triethylamine (142μl, 1.00 mmol) were combined in dichloromethane (4 ml). Methyl veleroylchloride (139 μl, 1.00 mmol) was added and the reaction mixture stirredat ambient temperature overnight. The reaction mixture was concentratedin vacuo and the resulting solid was dissolved in ethyl acetate, andwashed with aqueous sodium bicarbonate solution, water and brine. Theorganic layer was dried over magnesium sulfate and concentrated. 1:1dichlormethane/trifluoroacetic acid (3 ml) was added and stirred for 30minutes. The solution was concentrated in vacuum, then dissolved in DCM,(polystyrylmethylammonium bicarbonate was added, stirred for 4 hours.Resin was removed by filtration. Solvent was simply removed byevaporation.

Step 2:

1-(4-Amino-piperidin-1-yl)-4-methyl-pentan-1-one (133 mg, 0.67 mmol) andN,N-diisopropylethylamine (140 μl, 0.80 mmol) were dissolved indichloromethane (3 ml) and cooled to 0° C. Triphosgene (80 mg, 0.268mmol) in DCM was added and the mixture was stirred 20 minutes. Thesolvent was evaporated and the intermediate dissolved in THF (3 ml) with6-isopropylamino-1,3-benzothiazole-2-thiol (90 mg, 0.403 mmol) andheated at 60° C. overnight. The product was purified by preparative TLC(20% acetonitrile, 80% dichloromethane) to yield 68 mg (23%). ¹H NMR(CDCl₃) δ 0.85 (d, 6H), 1.22 (d, 6H), 1.45 (m, 3H), 1.8 (d, 1H), 1.95(d, 1H), 2.23 (m, 2H), 2.65 (t, 1H), 3.16 (t, 1H), 3.70 (m, 3H), 4.4 (d,1H), 4.8 (m, 1H), 7.14 (d, 1H), 7.22 (s, 1H), 7.39 (d, 1H); ESIMS: m/z447 (M−H).

Example 4 Preparation of4-[3-(2-Mercapto-benzothiazol-6-yl)-3-methyl-ureido]-piperidine-1-carboxylicacid propylamide

¹H NMR (CDCl₃) δ 0.95 (t, 3H), 1.01 (d, 6H), 1.5 (m, 2H), 1.8 (m, 5H),2.89 (t, 2H), 3.2 (t, 2H) 3.79 (m, 1H), 4.75 (m, 1H), 7.14 (d, 1H), 7.22(s, 1H), 7.39 (d, 1H); ESIMS: m/z 434 (M−H).

Example 5 Preparation of3-(1-Benzenesulfonyl-piperidin-4-yl)-1-(2-mercapto-benzothiazol-6-yl)-1-methyl-urea

¹H NMR (CDCl₃) δ 1.42 (m, 2H), 1.91 (m, 2H), 2.32 (t, 2H), 3.2 (s, 3H),3.62 (m, 3H), 7.15 (d, 1H), 7.23 (d, 2H), 7.55 (t, 2H), 7.6 (d, 1H),7.65 (d, 2H). ESIMS: m/z 461 (M−H).

TABLE 1 Examples

Examples R₁ CBM-000302130 PhCH₂OCO CBM-000302182 CF₃(CH₂)₃ CBM-000302198(CH₃)₂C(CH₂)₃ CBM-000302215 i-PrSO₂ CBM-000302216 p-n-Bu0-PhCOCBM-000302217 p-CF₃-PhCO CBM-000302218 PhOCH₂CO CBM-000302219p-CF₃-PhNHCO CBM-000302220 PhCH₂CH₂CO CBM-000302274 n-PrNHCOCBM-000302275 c-C₅H₉CH₂CH₂CO CBM-000302276 n-Bu-SO₂ CBM-000302277 PhSO₂CBM-000302278 p-CF₃O-PhSO₂ CBM-000302279 p-CF₃-PhSO₂ CBM-000302280m-CF₃-PhSO₂ CBM-000302282 BuOCO CBM-000302313 i-BuCH₂CO CBM-000302314n-PrCO CBM-000302316 CH₃OCOCH₂CH₂CO CBM-000302342 m-Cl-PhSO₂CBM-000302343 m-CN-PhSO₂ CBM-000302344 p-t-Bu-PhSO₂ CBM-000302348p-F-PhCO CBM-000302359 p-CH₃O-PhCO CBM-000302360 2-furanyl-COCBM-000302361 p-CF₃O-Ph CBM-000302368 p-i-PrO-PhSO₂ CBM-000302369m-F-PhSO₂ CBM-000302384 p-n-Pr-PhSO₂ CBM-000302386 m-CH₃O-PhSO₂CBM-000302387 p-i-Pr-PhSO₂ CBM-000302389 p-CH₃CO-PhSO₂ CBM-000302404p-Et-PhSO₂ CBM-000302405 PhCH2SO₂ CBM-000302406 3-Cl-4-F-PhSO₂CBM-000302407 p-CH₃O-PhSO₂ CBM-000302424 p-CN-PhSO₂ CBM-000302425p-F-PhSO₂ CBM-000302426 2-Cl-3-Br-5-PySO₂

TABLE I in vitro MCD inhibitory activities Examples Ki (nM)CBM-000302130 11.0 CBM-000302182 80.0 CBM-000302198 56.7 CBM-00030221519.1 CBM-000302216 88.1 CBM-000302217 42.3 CBM-000302218 7.4CBM-000302219 30.5 CBM-000302220 20.4 CBM-000302274 40.6 CBM-00030227522.4 CBM-000302276 0.7 CBM-000302277 2.8 CBM-000302278 104.6CBM-000302279 61.6 CBM-000302280 2.6 CBM-000302282 53.4 CBM-00030231323.5 CBM-000302314 58.5 CBM-000302316 99.6 CBM-000302342 0.7CBM-000302343 3.8 CBM-000302344 132.6 CBM-000302348 40.7 CBM-0003023597.2 CBM-000302360 24.0 CBM-000302361 87.2 CBM-000302368 624.3CBM-000302369 2.8 CBM-000302384 66.1 CBM-000302386 1.3 CBM-000302387124.6 CBM-000302389 84.3 CBM-000302404 27.5 CBM-000302405 7.8CBM-000302406 1.4 CBM-000302407 17.5 CBM-000302424 123.2 CBM-0003024251.4 CBM-000302426 19.3

TABLE II Glucose Oxidation of MCD inhibitors in Isolated Working RatHearts Examples MW GOX (%)^(a) DMSO 100 CBM-000302276 470.7 310CBM-000302280 558.7 683 CBM-000302281 462.6 190 CBM-000302218 484.6 236CBM-000302342 525.1 285 CBM-000302343 515.7 177 ^(a)Glucose oxidationrate relative to DMSO (100%)

1-3. (canceled)
 4. A compound according to claim 16, which is:4-[3-Isopropyl-3-(2-mercapto-benzothiazol-6-yl)-ureido]-piperidine-1-carboxylicacid benzyl ester;1-Isopropyl-1-(2-mercapto-benzothiazol-6-yl)-3-[1-(4,4,4-trifluoro-butyl)-piperidin-4-yl]-urea;1-Isopropyl-1-(2-mercapto-benzothiazol-6-yl)-3-[1-(4-methyl-pentyl)-piperidin-4-yl]-urea;3-[1-(4-Butoxy-benzoyl)-piperidin-4-yl]-1-isopropyl-1-(2-mercapto-benzothiazol-6-yl)-urea;1-Isopropyl-1-(2-mercapto-benzothiazol-6-yl)-3-[1-(4-trifluoromethyl-benzoyl)-piperidin-4-yl]-urea;1-Isopropyl-1-(2-mercapto-benzothiazol-6-yl)-3-[1-(2-phenoxy-acetyl)-piperidin-4-yl]-urea;4-[3-Isopropyl-3-(2-mercapto-benzothiazol-6-yl)-ureido]-piperidine-1-carboxylicacid (4-trifluoromethyl-phenyl)-amide;1-Isopropyl-1-(2-mercapto-benzothiazol-6-yl)-3-[1-(3-phenyl-propionyl)-piperidin-4-yl]-urea;4-[3-Isopropyl-3-(2-mercapto-benzothiazol-6-yl)-ureido]-piperidine-1-carboxylicacid propylamide;3-[1-(3-Cyclopentyl-propionyl)-piperidin-4-yl]-1-isopropyl-1-(2-mercapto-benzothiazol-6-yl)-urea;4-[3-Isopropyl-3-(2-mercapto-benzothiazol-6-yl)-ureido]-piperidine-1-carboxylicacid tert-butyl ester;1-Isopropyl-1-(2-mercapto-benzothiazol-6-yl)-3-[1-(4-methyl-pentanoyl)-piperidin-4-yl]-urea;3-(1-Butyryl-piperidin-4-yl)-1-isopropyl-1-(2-mercapto-benzothiazol-6-yl)-urea;4-{4-[3-Isopropyl-3-(2-mercapto-benzothiazol-6-yl)-ureido]-piperidin-1-yl}-4-oxo-butyricacid methyl ester;3-[1-(4-Fluoro-benzoyl)-piperidin-4-yl]-1-isopropyl-1-(2-mercapto-benzothiazol-6-yl)-urea;1-Isopropyl-1-(2-mercapto-benzothiazol-6-yl)-3-[1-(4-methoxy-benzoyl)-piperidin-4-yl]-urea;3-[1-(Furan-2-carbonyl)-piperidin-4-yl]-1-isopropyl-1-(2-mercapto-benzothiazol-6-yl)-urea;or1-Isopropyl-1-(2-mercapto-benzothiazol-6-yl)-3-[1-(4-trifluoromethoxy-benzoyl)-piperidin-4-yl]-urea;or a pharmaceutically acceptable salt thereof. 5-15. (canceled)
 16. Acompound having the formula:

wherein R₁ is —COR₄ or CONR₄R₅; R₂ is hydrogen; R₃ is isopropyl; R₄ ispropyl, 4,4,4-trifluorobutyl, pentyl, 4-methylpentyl, butoxy,methoxycarbonylethyl, cyclopentylethyl, phenethyl, phenoxymethyl,benzyloxy, furan-2-yl, or para-substituted phenyl where the substituentis fluoro, methoxy, butoxy, trifluoromethyl or trifluoromethoxy; and R₅is hydrogen; or a pharmaceutically acceptable salt thereof.
 17. Apharmaceutical composition comprising a compound of claim 16 and apharmaceutically acceptable carrier.